Immunoelectron microscopic study of the insoluble antigens of bone marrow cells in rats

An immunoperoxidase study of the localization of insoluble antigens was carried out on the rat bone marrow cells. The effect of different fixatives and inhibitors of endogenous peroxidase on the cell ultrastructure and the preservation of immunoreactivity of the cell antigens. The best results were obtained while fixing with 1% paraformaldehyde and 0.05% glutaraldehyde mixture added with 0.5 saponin and using 10% acetic acid as an inhibitor of endogenous peroxidase. Differences were found in the localization of antigens and the intensity of immunoperoxidase staining in cells of different lines of differentiation and degree of maturity.     

Immunoelectron microscopic studies of intermediate filaments in cultured cells

Indirect immunoferritin labelling has been used to show the localization of the 57 000 D cytoskeletal protein vimentin in intermediate filaments. Labelling could be shown using either detergent-extracted cytoskeletons prepared from unfixed cultured cells or cells subjected to light fixation and rendered permeable to antibodies by treatment with saponin. Immunoferritin and immunoperoxidase labelling methods were combined with stereo electron microscopy to describe the three-dimensional organization of the intermediate filament system in cultured cells of fibroblastic morphology. The filaments form a complex three-dimensional network throughout the cytoplasm and are frequently found to be associated with microfilament bundles near the upper and lower plasma membranes. The filaments also surround the nucleus and may therefore play a role in anchoring this organelle in the cytoplasm.     

Immunoelectron microscopic studies of type X collagen in endochondral ossification

Immunofluorescence and immunoelectron microscopy were used in conjunction with a monoclonal antibody to investigate the localization of type X collagen in the proximal tibial growth plate of 7-d-old chicks. This molecule was detected throughout the hypertrophic zone first appearing when chondrocytes exhibited hypertrophy: it was absent from the proliferative zone. Type X collagen was primarily associated with type II collagen fibrils as demonstrated by immunogold staining. Type X collagen was not concentrated in the focal calcification sites nor was it associated with matrix vesicles. These observations suggest that type X collagen may play a role other than that directly related to the nucleation of calcification.     

Immunoelectron microscopic studies of the ultrastructure of myosin filaments in Dictyostelium discoideum

We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4 degrees C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide. Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the microfilaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl2, but they were similar to those formed in the presence of 2 mM MgCl2, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.     

Immunoelectron microscopic studies on spore coat proteins of Bacillus megaterium

An immunochemical staining technique for the spore coat proteins of Bacillus megaterium ATCC 12872 was developed using colloidal gold as a second antibody. For reducing the non-specific immunogold binding and increasing the specific binding, the affinity-purified IgG was used as a first antibody. In sporulating cells at t10, gold particles were found not only in the spore coat but also in the mother cell cytoplasm, suggesting that some coat proteins were synthesized in the cytoplasm. Use of the specific affinity-purified antibody to 48K-protein demonstrated that this protein was one of the components of the outer coat.     

Immunoelectron microscopic (IEM) studies on glutaraldehyde-fixed renal specimen

Immunofluorescent microscopy (IF) has become an essential tool in the routine diagnosis of renal pathology. It is thought that glomerular IF patterns represent different forms of immunologically mediated glomerular lesions. However, in order to preserve antigenicity IF is usually done on frozen, unfixed specimen by light microscopy and it is difficult to locate target macromolecules in tissue with precision. In order to establish the precise location of such macromolecules in glomeruli in relation to the ultrastructure, we undertook an immunoelectron microscopic (IEM) study on renal biopsies. Percutaneously biopsied tiny specimen was fixed in glutaraldehyde and processed with protease. Using peroxidase-labeled antisera, a direct IEM was done. With this technique, target macromolecules corresponded well not only to electron-dense deposits but also to IF patterns. In one case of membranous nephropathy, immunoglobulin (Ig) was found to be outside of the glomerular basement membrane (GBM), while IgA was seen to be inside the GBM. For this same case, a similar localization of immunoglobulins was not revealed by IF. In conclusion, IEM is a useful technique that can broaden our knowledge of pathogenic mechanisms in glomerular disease.     

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts

Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double immunoelectron- microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and alpha- actinin with respect to these sites. (a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections. (b) FA sites were immunolabeled intracellularly for vinculin and alpha- actinin, with vinculin labeling situated closer to the membrane than alpha-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites. (c) CC sites were labeled intracellularly for alpha-actinin but not vinculin and were labeled extracellularly for fibronectin. (d) ECM sites were characterized by large separations (often greater than 100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both alpha-actinin and vinculin, the other for alpha-actinin alone. (e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and alpha-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Immunoelectron microscopic localization of tyrosinase in the mouse melanocyte

In the melanocyte, tyrosinase is known as the dey enzyme for melanin formation. Purified tyrosinase protein was prepared that was capable of oxidizing tyrosine. The localization of tyrosinase antigen in the melanocyte was studied using antiserum against tyrosinase. DOPA (L-3,4-dihydroxyphenylalanine)-reaction product and tyrosinase antigen were found on the same organelles i.e., premelanosomes, melanosomes, GERL, and Golgi vesicles. This result seems to suggest that it is cytochemically appropriate to use DOPA as the substrate of tyrosinase. It appeared that tyrosinase antigen was present as granule-like structures inside GERL cisterna and associated with its membrane.     

Immunoelectron microscopic classification of amyloid in renal biopsies

Recent classification of amyloidosis is based on the chemical type of amyloid protein involved. In this study, routinely embedded kidney biopsies from nine patients with generalized amyloidosis and renal involvement were tested by immunoelectron microscopy, using the protein A-gold technique, with a panel of antibodies against the following amyloid proteins: AA, A lambda, A kappa and AF. Among the antibodies, the anti-AA was monoclonal (mc1) and the others polyclonal. In all nine cases, only one type of antibody reacted with each amyloid type. Six cases were classified as AA and three cases as A lambda type. These classifications were in agreement with the clinical data and the results of serum and urine immunoelectrophoresis. The gold particles were always associated with amyloid fibrils. No reaction was evident when an amyloid type was stained by a non-corresponding antibody, or in the four control cases without amyloid. The results show that antigenic classification of amyloid is feasible on routinely processed ultra-thin epoxy sections by immunoelectron microscopy, and thus affords the possibility of retrospective studies.     

Immunoelectron microscopic localization of immunoglobulin in B-cell lymphomas

Subcellular localization of immunoglobulin (Ig) by immunoelectron microscopy was performed on 20 B-cell lymphomas of low- and high-grade malignancy. The efficiency in demonstrating Ig by pre-embedding technique depends on the antibodies used. F(ab')2 fragments of antibodies were more sensitive than both intact polyclonal and monoclonal antibodies in detecting cytoplasmic Ig. With immunoelectron microscopy Ig could be demonstrated in all cell types of B-CLL and LP-immunocytoma, even in some of the small lymphocytes in B-CLL. Thus, the presence of intracytoplasmic Ig has no diagnostic relevance in differentiating B-CLL from LP-immunocytoma. However, the amount of Ig in the tumor cells of LP-immunocytoma seemed to be greater than in B-CLL. Centrocytic lymphoma and centroblastic/centrocytic lymphoma could be differentiated by their different localization of Ig. In centrocytic lymphoma Ig was localized mainly on the surface membrane, whereas in centroblastic/centrocytic lymphoma moderate amounts of Ig could be detected in the rough endoplasmic reticulum and perinuclear space of the centroblasts and in roughly one third of the centrocytes. In malignant lymphomas of high-grade malignancy (ML centroblastic, ML immunoblastic, and ML lymphoblastic) Ig was localized mainly in the rough endoplasmic reticulum and sometimes in the perinuclear space.     

Immunoelectron microscopic localization of progesterone receptor in the chick oviduct

A peroxidase-anti-peroxidase (PAP) method using polyclonal anti-PR antibodies was used to localize progesterone receptor (PR) electron microscopically in the chick oviduct. The immunoreaction precipitate indicating PR was localized inside the nuclei of epithelial, glandular and stromal cells. In the estrogen withdrawn oviduct cytoplasmic immunoreaction precipitate was not seen. Inside the nucleus unoccupied PR was localized mainly like the heterochromatin. As visualized by the PAP technique, the localization of PR was not systematically changed after progesterone administration. In conclusion, we suggest that progesterone receptor in the chick oviduct is an intranuclear protein.     

Immunoelectron microscopic study of somatostatin biosynthesis in dog pancreas

The biosynthesis of somatostatin has been studied at the ultrastructural level in pancreatic islets by using rabbit antiserum against synthetic somatostatin. To document that the antiserum specifically bound preprosomatostatin, we have tested the ability of the antiserum to precipitate the product synthesized in vitro. Poly(A) enriched RNA isolated from catfish islets was translated in both the wheat germ extract and nuclease-treated reticulocyte lysate systems. It was found that the in vitro translation product, preprosomatostatin, could be recognized by the antibody against synthetic somatostatin. The morphological study was then performed by immunoelectron microscopy by using the Fab-peroxidase conjugate technique. In dog pancreatic islets, somatostatin immunoreactive reaction product was seen only in the delta cells. In these cells, they were detected on bound ribosomes, in the cisternae of the rough endoplasmic reticulum (ER) and Golgi apparatus, in the Golgi associated vesicles, and in secretory vesicles. These findings suggest that somatostatin precursor molecules are synthesized on bound ribosomes and discharged into the cisternae of the rough ER. They are then transported to the Golgi apparatus and transferred to the secretory vesicles for secretion. The different staining intensities in the secretory vesicles would suggest that the processing of the precursor molecules of somatostatin probably takes place in the secretory vesicles.     

Immunoelectron microscopic localization of calmodulin in corn root cells

Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.     

Immunoelectron microscopic localization of ubiquitin in hepatoma cells.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.     

Immunoelectron microscopic study of polyamines in the gastrointestinal tract of rat

Polyamines (PAs) are ubiquitous polycationic metabolites in the eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis. However, the subcellular localization of PAs has not yet been fully elucidated in a variety of cell types. In the present study, a pre-embedding indirect immunoperoxidase approach was used to define the fine structural localization of PAs in the gastrointestinal tract of rat, which was fixed with glutaraldehyde and the monoclonal antibody ASPM-29 specific for spermine (Spm) and spermidine (Spd). Examination by a transmission electron microscopy showed that the peroxidase end products were commonly and predominantly localized in the free and attached ribosomes of the rough endoplasmic reticulum (rER) in the active protein- or peptide-secreting cells, and in rapidly proliferating cells including the gastric chief cells, mucous neck cells, and intestinal crypt cells. The nuclei, mitochondria, and secretory vesicles were devoid of PAs. Of note is the new finding that PAs are also located even on the small number of ribosomes in the cytoplasm of the parietal cells and of the villus-tip cells, because these were the cell types that were found to be almost PA-negative at the light microscopic level. These results seem to be completely consistent with those recently obtained for rat neurons. Thus, the present study generalized the subcellular localization of PAs on the ribosomes, and demonstrated that PAs are one of the components of biologically active ribosomes, possibly in any type of cell, that are closely involved in the translation processes of protein biosynthesis.     

Sympathectomy alters bone architecture in adult growing rats

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.